In recent years, various biological activities of highly unsaturated fatty acids have attracted attention. For example, arachidonic acid is considered to be a precursor of prostaglandins, thromboxanes, prostacyclins, leucotrienes and the like that have biological activities such as uterine contraction and relaxation, vasodilation, and hypotensive activity. These substances have been under intensive research, together with docosahexaenoic acid, in recent years, as elements essential for the development of babies and infants. Various foods, cosmetics, and animal feeds to which a lipid containing highly unsaturated fatty acids such as γ-linolenic acid, dihomo-γ-linolenic acid, and eicosapentaenoic acid in addition to arachidonic acid and docosahexaenoic acid have been added are also attracting attention, and some of the products to which highly unsaturated fatty acids has been added are commercially available.
Accordingly, methods of efficiently producing these highly unsaturated fatty acids also are being studied intensively.
For example, methods of efficiently producing arachidonic acid, dihomo-γ-linolenic acid, γ-linolenic acid, or eicosapentaenoic acid by fermentation have been developed using microorganisms belonging to, for example, the genus Mortierella, specifically the subgenus Mortierella, that are known to produce highly unsaturated fatty acids such as arachidonic acid, dihomo-γ-linolenic acid, γ-linolenic acid, and eicosapentaenoic acid (Japanese Unexamined Patent Publication (Kokai) No. 63-44891, Japanese Unexamined Patent Publication (Kokai) No. 63-12290, Japanese Unexamined Patent Publication (Kokai) No. 63-14696, Japanese Unexamined Patent Publication (Kokai) No. 5-91887, and Japanese Unexamined Patent Publication (Kokai) No. 63-14697). There is also known a method of producing mead acid using a mutant strain in which Δ12 desaturation activity is decreased or missing, said strain being obtained by effecting mutation to microorganisms belonging to genus Mortierella subgenus Mortierella (Japanese Unexamined Patent Publication (Kokai) No. 5-91888).
Thus, the production of a lipid containing highly unsaturated fatty acids using microorganisms that produce said fatty acids is increasingly becoming a major source of highly unsaturated fatty acids. These microorganisms have a property of not only using highly unsaturated fatty acids they produce as constituents of the cell membrane but also accumulating highly unsaturated fatty acids as fat and oil (triglycerides) containing them as constituent fatty acids in the cell. By utilizing the fats and lipids accumulated in the cell, high productivity of highly unsaturated fatty acids have come to be secured.
In this conventional production method, the amount of fat and oil obtained per culture was a product of the cell mass of the microorganism obtained by culturing and the amount of fat and oil produced per cell, and thereby how to increase the cell mass and the amount of fat and oil per cell was a challenge to be solved in order to attain enhanced production of fat and oil. Research has so far revealed that the selection of culture conditions permits increases in both of the above to a certain extent, but there are certain limits to each of them with increases in the cell mass being limited by physical factors such as the volume of the culture tank and increases in the amount of fat and oil per cell being limited by physiological factors of the microorganism used.
On the other hand, when it is desired to utilize a lipid produced by and accumulated in the cell of the microorganism, it is necessary to collect the cells after culturing, process the cells with a mill etc., to disrupt the cell membrane, and then to extract the lipid accumulated in the cell.
If substances produced by a microorganism could be secreted outside of the cell instead of being accumulated in the cell, the physiological burdens placed on the microorganism by the substances produced can be alleviated and thus the microorganism can continue to produce the products; in the isolation and extraction of microbial products as well, extraction from the culture only is needed, which provides an advantage that treatment becomes simplified and microorganisms can be continuously processed as they remain alive.
Based on the above, efforts to extracellularly secret a lipid accumulated in the cell have recently been made by Sakuzo Fukui et al. (BIO INDUSTRY 12: 36-46 (1995)). Sakuzo Fukui et al. conducted research on the production and secretion of lipids by microorganisms to develop novel biological fuel that can replace fossil fuel, and have successfully bred genus Trichosporon yeast to convert sugar and n-alkanes to lipids and to secret them outside of the cell. They further demonstrated that the constituent fatty acid species of extracellularly secreted triglyceride (TG) are oleic acid, palmitic acid, linoleic acid, and stearic acid. However, it has a drawback that the TG that is directly secreted outside of the cell is incorporated into the cell again and metabolized.
However, microorganisms that have an ability to produce lipids containing unsaturated fatty acids that have 18 carbons and three or more double bonds or 20 or more carbons and two or more double bonds, and that have a property of extracellularly secreting the produced lipid, or microorganisms that have an ability of producing an unsaturated fatty acid-containing lipid, and that secrete the produced lipid encapsulated in small vesicles instead of directly secreting them at the time of extracellular secretion thereof, are not known.
In a study on γ-linolenic acid production by molds described in “Microbially produced fatty acids and their uses”, Osamu Suzuki, Fragrance Journal 1989 (6), pp. 67-75, it is reported that a surfactant was added to a culture medium of a microorganism of the genus Mucor to allow the leakage of some of lipids outside of the cell. However, it relates to methods of artificially destroying the cell membrane to allow lipids accumulated in the cell to leak out of the cell, and does not utilize the ability of the cell per se to extracellularly secrete lipids produced in the cell.
Thus, there is a need to find microorganisms that have an ability of producing a lipid containing unsaturated fatty acids that have 18 carbons and three or more double bonds or 20 or more carbons and two or more double bonds, and have a property of extracellularly secreting the produced lipids, or microorganism that have an ability of producing an unsaturated fatty acid-containing lipid, and that have a property of extracellularly secreting the produced lipids encapsulated in small vesicles, and to develop methods of efficiently producing lipids that contain unsaturated fatty acids using said microorganisms.
Incidentally, in order to find microorganisms that have novel abilities, the development of an efficient screening method for microorganisms having such abilities is a prerequisite. In the above breeding of genus Trichosporon yeast that converts sugar and n-alkanes into lipids and extracellularly secrete them, the following screening method was adopted. Thus, yeast colonies that appeared on an agar plate medium (YPD medium etc.) are UV-treated (15 watts at a distance of 30 cm for 15 minutes) (this treatment is intended to suppress the dispersion of colony cells during layering treatment and not to induce mutation), the UV-treated colony plates are layered with a YPD soft agar medium containing 105 cells of a test strains, and are then cultured at 28° C. for 2 days. As the test strains, the A-1 strain and the ole-1 strain having auxotrophy for saturated fatty acids and unsaturated fatty acids, respectively, are used and those colonies giving a larger halo (the micro colony ring of the test strain) around them are selected as lipid-secreting strains. The selection of lipid-secreting strains employs two media: a soft agar medium containing or not containing lipase.
This method has a major drawback that although it can be applied to screening of yeast for which layering is possible, it cannot be applied to screening of microorganisms for which layering is impossible. There is additional disadvantage that the assay method are complicated.
Thus, in order to find microorganisms that have an ability of producing a lipid containing unsaturated fatty acids that have 18 carbons and three or more double bonds or 20 or more carbons and two or more double bonds, said microorganisms having a property of extracellularly secreting the produced lipids, there is a need for the development of a screening method that permits simple and efficient screening of said microorganisms and that can be applied to various microorganisms, and the development of a new screening method for finding microorganisms that can secrete the produced lipids encapsulated in small vesicles at the time of extracellular secretion of the produced lipids.